Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is an important intermediate to synthesize a wide variety of secondary metabolites with potential biological activities. Recently, we engineered a biosynthetic pathway in Pseudomonas chlororaphis GP72 and optimized the batch-fermentation conditions to synthesize DHHA. Herein, we developed a proficient method for the separation and purification of DHHA from the culture extract of recombinant Pseudomonas chlororaphis GP72 by applying macroporous cation-exchange resins. The performance and absorption characteristics of six resins namely D001, HZD-2, JK006, HD-8, HZ016, and 732 were evaluated. Notably, HD-8 resin displayed the pronounced adsorption and desorption capacities for DHHA as compared to others and its equilibrium adsorption data were well-fitted to the Langmuir isotherm. Moreover, HD-8 column chromatography-based dynamic adsorption and desorption tests were carried out to standardize the operational parameters for DHHA separation and purification. Under the optimized separating conditions, the average adsorption capacity and desorption ratio of DHHA was 40.3 +/- 0.7 mg/g dry resin and 86.7 +/- 2.8%, respectively. The purity and recovery of DHHA was recorded to be 95.3 +/- 1.2% and 83.2 +/- 2.3%, respectively. Taken together, the HD-8 resin appeared as a promising adsorbent for high-level separation and purification of DHHA from the complex media. In conclusion, this method displays a high potential for separating and purifying DHHA and other bioactive compounds in high yield from the fermentation broth.